1. Field of the Invention
This invention relates to the field of biophysics and medicine, in particular to pharmaceuticals for the treatment of blood losses, hypoxia and ischaemic conditions and also for improving the blood oxygen transport and the preservation of isolated perfusing organs and tissues.
2. Discussion of Related Art
List of abbreviations, references, units and termsSurfactantssurface-active substancesP-268, F-268Proxanol 268, Pluronic 268PFDperfluorodecalinePFMHPperfluoromethylcyclohexylpiperidinePFOBperfluorooctylbromideFlorganic fluid which represents a mixture ofperfluorotripropylamine and its coproducts, cis- and trans-isomers: perfluoro-1-propyl-3,4-dimethylpyrrolidone andperfluoro-1-propyl-4-methylpiperidine.PFCsfluorocarbons, fluorocarbon compoundsPFTBAperfluorotributylaminePFTPA (PAF-3)perfluorotripropylamineSoya-Psoya phospholipidsEgg-Pegg phospholipidsnwavelength exponentCvvolumetric content of fluorocarbons in emulsion (ml/dl)aaverage particle sizeλwavelengthIpreactogenity index
The success in developing infusion media, which contain emulsions of fluorocarbon compounds, depends largely upon the physical-chemical properties of selected PFCs and emulsions based on these PFCs and also upon the production method.
PFCs for medicinal purposes represent fluorocarbon compounds of different classes. Externally, these are clear, colorless and odorless liquids with a very high density, approximately twice as heavy as water. An abnormally strong C—F bond (485.6 KJ/mol) leads to the fact that the intermolecular forces of these compounds are very weak. Weak intermolecular forces are manifested in their abnormally strong ability to dissolve gases, amongst them also blood gases.
The PFCs are characterized as a result of the strong C—F bond by chemical inactivity. They dissolve in water with difficulty and do not form the metabolic basis in organisms. The chemical inactivity of the PFCs cannot be equated to a biological inactivity. With intravenous injection of the emulsions on a PFC basis, these emulsions are retained in organs and tissues, the dwell time being dependent upon the nature of the PFC and the dose of the emulsion.
Investigations into the biological properties of perfluorinated compounds of different classes show that the elimination rate depends upon a series of connected physical-chemical parameters, namely upon the structure and the molecular weight, the boiling temperature, the vapor pressure and the critical dissolving temperature in hexane (Tcritical) Tcritical is that temperature at which the same volumes of the compound which is to be examined and of hexane mix. Tcritical is considered as a value of the relative PFC solubility in lipids, which value characterizes the rate of passage into membranes. The lower Tcritical is, the better the compound dissolves in lipids and the more rapidly it is eliminated from the organism. In Table 1, physical-chemical parameters are indicated which serve as selection criteria of PFCs for medicinal application.
TABLE 1Values for the critical solution temperature in hexane (Tcritical),the vapor pressure (P) and the half-decomposition time (t1/2)of different compounds [1].P,Half-mmdecompositionMolarQStime t1/2Perfluorinated compoundsweightTcritical(37°)24 hoursbicyclo[4.3.0]nonane41213334decaline46222127decahydroacenaphthene5242427N-(4-59538160 (90)methylcyclohexylpiperidine)1-propyl-2-methylpiperidine483351924tripropylamine521431765tributylamine671611900dihexylether652592500
From the above data a strong correlation between Tcritical and t1/2 can be seen. This correlation is not observed for the vapor pressure. To a great extent Tcritical and the molecular weight are interconnected. An optimal molecular weight for PFCs is the range between 460 and 520. Overall, all the offered selection criteria for medicinal PFCs are not mutually contradictory, but have a qualitative character. Nowadays, researchers who are involved in the development and examination of perfluorocarbon emulsions are directing their attention to a relatively restricted number of compounds. In Tables 2 and 3, the structural formulae and the physical-chemical main properties of the most widespread PFCs are indicated.
TABLE 2Structural formulae of the most widespread and promising PFVsperfluorodecalineperfluorotripropyl-perfluorotributylamine(PFD)amine (PFTPA)(PFTBA)mol. wt. 462mol. wt. 521mol. wt. 671  perfluorotrimethyl-perfluoromethyl-perfluoromethylcyclohexyl-bicyclononaneisoquinolinepiperidinemol. wt. 562mol. wt. 495mol. wt. 595  perfluorooctyl-bis-bis-fluorohexylethenebromide (PFOB)perfluorobutylethene(F-66E)CF3—(CF2)6—CF2Br(F-44E)C6F13—CH═CH—C6F13mol. wt. 499C4F9—CH═CH—C4F9mol. wt. 664mol. wt. 464
When examining primary biological properties of different PFCs, an important requirement is formulated: the absence of non-identifiable admixtures. Admixtures with unknown properties can distort the true behavioral picture (retention in organs, toxicity, influence on different systems of the organism) of the basic substance when injected intravenously.
TABLE 3Physical-chemical properties of PFCs which form the basis of medicinalpreparations.PerfluorodecylbromidePropertiesPFDPFTPAPFMHPPFOB(PFDB)StoichiometricC10F18C9F21NC12F23NC8F17BrC10F21BrformulaMol. weight, g/mol462521595499599Boiling temperature,142131168143180° C.Vapor pressure,12.718.02.010.51.5mm QS (37° C.)Critical solution224440−207temperature, (Tcritical)° C.Oxygen solubility40454053—ml/100 ml (vol.-%),(37° C.)Half-decomposition76590(60)440time t1/2Note:PFD/PFTPA are the basis of the preparation Fluosol-DA;PFD/PFMHP for the preparation Perftoran;PFOB/PFDB for the preparation Oxygent.
Liquid PFCs are poor solvents for various water-soluble, biologically active substances. For this reason, the PFCs for application as oxygen transport media are dispersed in an aqueous emulsifier solution until a finely distributed emulsion is obtained.
The ability of PFCs to exchange gases is determined according to the total oxygen content in the emulsion. The oxygen concentration is subject to Henry's Law and is directly proportional to the oxygen pressure. The principle of the physical solubility of the gases in the PFCs extends also to the perfluorocarbon emulsions. The oxygen quantity dissolved in the emulsion depends upon the fluorocarbon phase and not upon the particle size, i.e. the oxygen quantity dissolved in the fluorocarbon emulsion approximates to the values calculated by a summation of the gas quantity values of each phase (oxygen quantity in the aqueous phase plus oxygen quantity in the PFCs). The content of inert gases in the mixture of PFC and plasma is also subject to the summation law of the gas quantity of each phase. Hence, the content of each gas in the emulsion can be calculated according to physical laws of the solubility thereof due to the partial gas pressure and volume ratio of the fractions PFC/H2O. This means that the oxygen content in perfluorocarbon emulsions raises as its partial pressure or its tension (pO2) and the proportion of the fluorocarbon phase raise.
The specific (functional) effect of each preparation when injected into the body is determined by the compatibility of the preparation, which is determined by the LD50 value and also by the lack of side-effects which appear mainly as the reactogenity. The size of the LD50 value for PFC emulsions depends greatly upon the particle size. The average particle size must not exceed 0.2 μm. An increase in the proportion of large particles (average size over 0.4 μm) of 3% to 10% reduces the LD50 value for the mentioned emulsions by a factor of two. Detection of a possible reactogenity of the perfluorocarbon emulsions is one of the most difficult problems which has to be solved when developing a pharmaceutical form based on the perfluorocarbon emulsions for intravenous injection. When using a reactogenity preparation, an allergic reaction can develop in humans which manifests itself in different ways, from slight reddening of the skin to anaphylactic reaction with cessation of breathing and cardiac arrest.
Most researchers are of the opinion that for the most part reactogenity depends upon the nature of the emulsifier which is used for the dispersion of the fluorocarbon basis of the emulsion and which forms a (superficial) absorption layer around the particles. It is believed that the reactogenity of first generation emulsions was caused by the non-ionic block polymer of oxyethylene and polyoxypropylene, Pluronic F 68 (F-68), and that exchange thereof by natural phospholipids completely solves the reactogenity problem. This opinion is not completely correct, because fat emulsions, despite stabilisation by natural phospholipids, possess reactogenity. The reactogenity of the perfluorocarbon emulsions cannot simply be eliminated by the use of phospholipids as emulsifier and stabilizer. In actual fact, it emerged that the reactogenity of the PFC emulsions is effected above all by the surface properties of the emulsified particles, i.e. by the state of the emulsifier layer which stabilizes the particles. In addition to the chemical structure, the nature of the surfactant molecules and the key parameters which determine both the stability of the disperse system and possible secondary reactions, the binding strength of the surfactants with the oil nucleus of the emulsion particles, the position of the molecules on the surface, the density of the packing thereof, the prevalence of the absorption properties relative to proteins and other biologically active molecules which are situated in the bloodstream and finally the size of the emulsion particles play a part. The last parameter should in particular be mentioned. A decrease in the average particle size of the emulsion in the preparation Perftoran, which is only stabilized by the block copolymer polyoxyethylene and polyoxypropylene, Proxanol 268 which is the nearest prototype to F-68, leads to a rapid reduction in the secondary reaction. It is clear from this that in the development, formulation and production method of the emulsions, superficial phenomena (interaction of two heterogeneous systems, emulsion and blood or plasma) play a decisive role in the behavior of the intravenously injected emulsion. The composition of the oil nucleus and also the surfactant which cooperates with the latter should hereby be selected experimentally and also the tenability of the technology used should be tested.
When developing the perfluorocarbon emulsion according to this invention for medicinal purposes and the production method, each formulation and each technological element was examined for biological effect by an animation model. It is known that the reactogenity reaction of rabbits, when injected with perfluorocarbon emulsions, is expressed by a rapid decrease in neutrophilic leucocytes in the peripheral blood. When evaluating possible reactogenity of the perfluorocarbon emulsions, a reactogenity index Ip is used in tests, which is calculated according to the formula Ip=Ck/Cv in which Ck and Cv designate neutrophiles in % relative to the initial level in the control and test group. If after 5 and 20 minutes Ip is less than 3, then the reactogenity probability is minimal [3].
Different methods for producing perfluorocarbon emulsions are known. Oil in water emulsions, which include perfluorocarbon emulsions and in which the perfluorocarbon basis is an oil phase, are produced at a high cost in energy. Comminution of the oil phase is implemented by ultrasound or mechanically.
Under the effects of ultrasound, a dispersion is implemented by frictional forces with intense local pressure change which has two causes. First, local compression and expansion alternate in the liquid with the passage of waves. Second, cavitation occurs, i.e. formation and collapse of cavities which are filled with the gases dissolved in water. The energy and the force of the ultrasound effect which are necessary in order to produce a sub-microemulsion are so large that, in addition to the dispersion, the C—F bond is broken. As a result, highly toxic concentrations of the F ions, approximately 3-5 mmol, appear in the aqueous phase of the emulsion. An emulsion with such a high concentration of F− ions cannot be used for blood replacement or for preserving perfusing organs. It is necessary to free it of the excess of F− ions by passage through an ion exchange resin. The second disadvantage of an emulsion dispersed by ultrasound is in an exceptionally high dispersion range because, with an average particle size of 0.1 μm, a large particle proportion can be found to be over 0.4 μm and under 0.01 μm in size.
A mechanical dispersion by shaking or intense agitation permits emulsions which are only coarsely dispersed to be obtained, with a particle size of over one millimetre which is not acceptable for biomedicinal application. In order to produce finely distributed emulsions forced passage of the substance of the disperse phase through fine holes into the dispersion medium under high pressure (extrusion) is used, as a result of which the liquid jet is broken up into droplets. The dispersion is effected by the pressure gradient and hydraulic frictional forces. The emulsions are normally produced in high pressure homogenisers. Stabilization of the obtained emulsions is achieved with the help of surface-active substances or emulsifiers. The stabilizing effect of these substances is explained by two causes: first by the reduction in excess surface energy between the phases or by the reduction of the surface tension and second by the formation of a structural, mechanical barrier (absorption layer) which ensures the stability of the particles and prevents contact or adhesion or agglomeration of the particles.
Amongst many surfactants, only a few fulfil the requirements for applicability to the production of preparations for intravenous injection (Table 4).
TABLE 4Common surface-active substances for the production of perfluorocarbonemulsionsDescriptionStructural formulaBasic parametersProxanol 268 (Pluronic F-68)Synthetic blockcopolymer, mol.wt. ~13000 (P-268) and ~9000 (F-68), x = number of chain members of the ethylenepolyoxide block, y = number of chain members of the propylenepolyoxide block. Readily soluble. phospholipidsNatural compound. R1 and R2 are different chains of the fatty acids. (Egg yolk)R3═N(CH3)3Mol. wt. 760-870.lecithinPractically insoluble inwater
At the moment, mainly two emulsifiers are used to produce perfluorocarbon emulsions, namely Proxanol-268 (Pluronic F-68) and natural phospholipids (egg and soya phospholipids etc.).
The Proxanol structure does not correspond to the characteristic molecular properties of water-soluble surfactants which have a polar head (hydrophilic part) and a non-polar tail (hydrophobic part). In the case of Proxanol, the hydrophilic molecular character is determined by two polyoxideethylene chains, the hydrogen bonds being formed with H2O molecules. Methyl groups of polypropylenepolyoxide make lipophilic properties of its molecule a prerequisite. The ratio of the polyoxideethylene/polyoxidepolypropylene blocks for F-68 and P-268 is the same on average and is 80:20. The stabilizing effect of these emulsifiers is effected mainly by the steric effect of the protective film which is formed by the surface-active molecules around the fluorocarbon particles. The largest part of the surfactant molecules, in addition to the surfactants bonded in the absorption layer, thereby forms various micellar structures in the aqueous phase, including those which are free of fluorocarbon compounds. Between the surfactant molecules in the absorption layer and in the micells of the aqueous phase, a dynamic equilibrium is present which, on the one hand, is required for stabilisation of the absorption layer and, on the other hand, disturbs the density of the molecular packing of the surfactants in the absorption layer during long-term storage.
The phospholipids represent a mixture of compounds of natural origin, the general structure of which is indicated in Table 4. Phospholipids are water-insoluble and, at the same time, poorly lipophilic active substances with respect to different fluorocarbon compounds although they are partially dissolved by PFD and PFTPA in the double layer of the phosphatidyl choline particles. The cooperation of the phospholipids and fluorocarbon compounds in the aqueous phase has a double character. It is possible to include fluorocarbon compounds in the lamella structure of the phospholipids and/or to form monolayers of the phospholipids which are connected irreversibly to the particle surface. Non-homogeneous particles are possible in emulsions comprising fluorocarbon compounds and phospholipids, i.e. particles which are covered with a protective layer comprising phospholipids and free of phospholipids. This non-homogeneity can be attributed to production particularities and/or phospholipid excess relative to the fluorocarbon phase.
For finely distributed emulsions, the determining mechanism for reducing fineness (particle coarsening) is isothermic or molecular substance distillation of the disperse phase from small to larger particles by diffusion of the molecules of fluorocarbon compounds through a dispersion medium. This process is called “Ostwald ripening” of the emulsion or “recondensation”. The driving force of this process is an increased pressure of saturated vapor over smaller particles in comparison to larger. In this case, an important parameter is also the level of solubility of fluorocarbon compounds in the aqueous medium. Prevention of recondensation can be of crucial importance for obtaining a resistant aggregate state of the perfluorocarbon emulsions, i.e. obtaining the fineness and individuality of the particles. The main routes to destabilization, namely molecular diffusion and a less significant flocculation and coagulation, are characteristic both of relatively dilute emulsions, in which the fluorocarbon phase is below 20% by volume, and of more highly concentrated emulsions in which the fluorocarbon phase is 50% by volume.
The stabilization routes of the perfluorocarbon emulsions are known. The basic principle of stabilization of colloid systems means prevention of their decomposition mechanisms. Addition of sugar and coemulsifiers with a negative charge (minority components of the phospholipids) in emulsions on a PFC/phospholipid basis prevents flocculation of the particles by changing the spatial interaction of the surfactant molecules in the absorption layer and also by increasing the electrostatic repulsion force between the particles.
Reducing the main decomposition process of the perfluorocarbon emulsions, which is caused by molecular diffusion, is achieved by addition of a second less water-soluble component (additional fluorocarbon compound) to the fluorocarbon basis which has a higher boiling temperature and slows down this process.
The principle of this stabilization is used in the development of the preparations Fluosol-DA, Perftoran and Oxygent. The compiled data are represented in the following Table 5 according to the composition and the physical-chemical properties of the mentioned preparations.
TABLE 5Compiled data according to the composition of the preparations Fluosol-DA(Japan), Perftoran (Russia) and Oxygent (USA)/2/.Concentration (% by vol./wt.)OxygentIngredientsFluosol-DAPerftoranAF0104AF0143AF0144Perfluorodecaline (PFD)1413———Perfluorotripropylamine6————(PFTPA)Perfluoromethylcyclohexyl-—6.5———piperidine (PFMHP)Perfluorooctylbromide——908758(PFOB)Perfluorodecylbromide———32(PFDB)Pluronic F-68 (Proxanol-268)2.724———Phospholipids0.4— 45.43.6Potassium oleate0.032————Buffer substanceCO3−2CO3−2PO4−3PO4−3PO4−3Bivalent cations++———
In the first two preparations, the fluorocarbon compounds perfluorotripropylamine and perfluoromethylcyclohexanepiperidine are added as supplements with a higher boiling temperature and less water-soluble to perfluorodecaline which has the greatest proportion of the oil phase. Water-soluble Pluronic F-68 with phospholipid supplement (Fluosol-DA) or its prototype Proxanol-268 (Perftoran) is used as emulsifier. They differ little from each other according to their physical-chemical properties. They belong to preparations of the first generation, the general disadvantage of which resides in the fact that, because of inadequate stability, they must be stored frozen. Perfluorodecylbromide, which has a higher boiling temperature and is less water-soluble, is added to the fluorocarbon basis of Oxygent (perfluorooctylbromide). The advantage of Oxygent which belongs to the second generation is determined by storage in the non-frozen state. Furthermore, perfluorooctylbromide, which is the fluorocarbon basis of the preparation is eliminated rapidly from the organism almost at the same rate as perfluorodecaline (corresponding to t1/2˜4 and 7 days).
Oxygent is a trade name of infusion media which are somewhat different with respect to composition.
The emulsifier not only contributes to lowering the superficial intermediate phase tension in the H2O/PFC system which is required for fineness. A change in the emulsifier nature can influence the rate of the molecular diffusion. Fluorinated surfactants, which contain a fluorinated, hydrophobic and a non-fluorinated hydrophilic part in their molecule, are considered to be promising for the future. Great success in the synthesis of fluorinated surfactants for fluorocarbon compounds was achieved recently by French chemists [4]. The general structure of synthetic, fluorinated surfactants represents a combination of a perfluorinated chain and a polar head. A hydrocarbon chain is used as binding link of these elements. The polar head is selected from natural substances or derivatives thereof. Fluorinated surfactants, which contain alcohols or sugar derivatives as polar head, have a synergy with Pluronic F-68. The use of phospholipids, sugar phosphates or phosphatidyl choline in fluorinated surfactants as polar head increases the stability of the fluorocarbon emulsions which contain natural phospholipids as emulsifiers. A new class of mixed, fluorinated surfactants was proposed for stabilization [4]. The molecules of this class of fluorinated surfactants represent a block of two linear components, namely a hydrocarbon component and a perfluorinated component. The general formula of these compounds is as follows:CnF2n+1CmH2m+1 or CnF2n+1CH═CHCmH2m+1 
The inventors name these molecules “dowel” which means literally “spring” or “connection element”.
The opinion prevails that molecules of fluorinated surfactants with a general, linear RH—RF structure play the role of a strengthening element, the hydrocarbon end of which enters into the lipid film which covers the perfluorocarbon particles and the other fluorinated end of which enters into the oil phase, i.e. that the RH-RF molecules improve the adhesion properties of the surfactant surface layer.
Now, perfluorodecaline and perfluorooctylbromide are the most accepted compounds for producing biomedicinal emulsions for the reason that they are eliminated rapidly from the organism in comparison to other fluorocarbon compounds.
Patents [5, 6] are known in which compositions of blood replacement agents are described, the fluorocarbon basis of which represent mixtures of two (perfluorodecaline/perfluoromethylcyclohexylpiperidine or perfluorodecaline/perfluorotributylamine or perfluorooctylbromide/perfluoromethylcyclohexylpiperidine), of three (perfluorooctylbromide/perfluorodecaline/perfluoromethylcyclohexylpiperidine or perfluorooctylbromide/perfluorodecaline/perfluorotributylamine) or even of four fluorocarbon compounds (perfluorooctylbromide/perfluorodecaline/perfluoromethylcyclohexylpiperidine/perfluorotributylamine) in a different ratio. These mixtures disperse by the water-soluble emulsifier Proxanol P-268. The use of this emulsifier does not make it possible to store the mentioned mixtures at positive temperatures. Furthermore, these emulsions, after thawing, have a limited storage duration at +4° (at most 1 month). That is their main disadvantage.
Emulsions with fluorinated surfactants are patent-protected. The known micro-emulsions containing fluorinated surfactants [7] have no practical application as infusion medium more for the reason that they are not sufficiently stable in vivo. Another composition of perflurocarbon emulsions, which are produced by mixed, fluorinated surfactants, is known, containing a fluorophilic part and a lipophilic part in the molecule [8]. These emulsions maintain in fact the mean particle average at positive temperatures but only within 3 months.
A patent [9] is known, in which a 10% fat emulsion of liposyn serves to produce emulsions as phospholipid source. Three groups of fluorocarbon compounds are patent-protected as fluorocarbon basis. Belonging to the first group are perfluorocycloalkanes or perfluoroalkylcycloalkanes (amongst those perfluorodecaline, perfluoromethyldecaline, perfluoroperhydrophenanthrene inter alia). The second group comprises perfluoroalkyl-saturated, heterocyclic compounds. The third group comprises perfluorinated, tertiary amines and perfluorotributylamine, perfluorotripropylamine inter alia. Perfluorooctylbromide also belongs to the applicable fluorocarbon compounds. However, it is still not possible to produce a stable perfluorodecaline emulsion with the help of the 10% liposyn. Its maximum storage duration is 25 days.
In a further patent [10], egg phospholipids are used for emulsion production. The proportion of the fluorocarbon phase changes within a large range of 10 to 50% by volume and that of the phospholipids from 0.5 to 7% by weight. As oil phase, only one of the PFCs from the broad class of compounds is selected and used in the patent, namely the perfluorohydrophenanthrene group with fluorine atoms from 1 to 24, perfluorodecaline, perfluorooctylbromide, perfluoromethyladamantane and perfluoroperhydrophenanthrene.
The main focus in both mentioned patents is on methods for preserving different organs and systems by the use of produced fluorocarbon emulsions. At the beginning of physiological tests, emulsions are mixed with crystalloid solutions and/or oncotic active substances (albumin, hydroxyethyl starch). The proposed emulsions in fact belong to emulsions of the second generation but have a substantial disadvantage. In both patents, examination results for emulsion stability, i.e. maintaining the particle size with long-term storage (over a month), is not indicated. The two just mentioned patents [9, 10] are regarded here as prototypes.
The closest prototype to the emulsion according to this invention is the emulsion mentioned under [11]. This emulsion, regarded as prototype, belongs to the second generation and contains a rapidly eliminated fluorocarbon compound in the quantity of 40 to 50% by volume and a perfluorinated supplement of a higher-boiling compound of 5 to 10% by volume. As a rapidly eliminated fluorocarbon compound, perfluorodecaline or perfluorooctylbromide (main component) is used and, as supplement, perfluoromethylcyclohexylpiperidine. The emulsifier is egg or soya phospholipid.
The perfluorocyclohexylpiperidine stabilises the emulsion, reduces the rate of molecular diffusion (recondensation) of the main components (perfluorodecaline or perfluorooctylbromide) and is used to produce emulsions of a different composition, namely Perftoran. The main disadvantage of the emulsion known from patent [11] is a relatively large particle average above 0.2 μm.